Genetic transformation of plants is gradually beginning to play an important role in modern agriculture. Attempts are made to introduce heterologous DNA into plants in order to increase their resistance to viral infection, acquire or increase resistance to various herbicides, modulate ripening or decay times, increase the nutritional value of various plant products, bring them to produce pharmaceuticals, and produce various other chemical and biological molecules.
Commercial production of transgenic compounds in bacterial, yeast and mammalian cell systems is often beset by high capital investment costs in fermentation equipment and the necessity to eliminate prion or microplasmal components from the purified product. Recently, production of heterologous proteins and peptides (e.g., α-amylase, antibodies, enkephalins, human serum albumin) has been achieved in plants (Pen et al., 1992, Miele, 1997). Potential advantages of transgenic plants systems are: lowered production costs of biomass and a reduction in the biohazard of contaminants in downstream processing of the products. Transgenic plants may thus be superior bioreactors for bulk enzymes in industry, purified products in medicine and orally active pharmaceuticals.
In order to transform plants to produce a desired product, the relevant gene, once identified and cloned, has to be introduced into the plant of interest so that the resulting plant is capable of passing the gene to its progeny. The methods of introduction proposed for this purpose include electroporation, microinjection, microprojectile bombardments, liposome fusion, Agrobacterium mediated transfer, and many others.
One of the most commonly used transforming vectors is Agrobacterium, which is a genus of plant pathogenic bacteria of the family Rhizobiaceae, which does not fix free nitrogen and usually produces gall and hairy roots in infected cells. Heterologous DNA is introduced into the Agrobacterium and through a process of transfection wherein genetic material from the Agrobacterium enters the plant's cell, genetic transformation of the plant takes place (Armitage et al., 1992). Agrobacterium infects primarily dicotyledonous plants and infects monocotyledonous plants only at a very low yield (Armitage et al., 1992).
One attempt to transform monocotyledonous plants was by a particle gun wherein the heterologous DNA is delivered by air or by helium into the plant or plant cell to be transformed. This technique has two main disadvantages: first, it is quite difficult to target the DNA particles to the meristematic zone wherein, for certain plants such as those of the family Lemnaceae, the transformation should take place in order to enable regeneration therefrom of a full transformed plant; second, even if the DNA particle enters the cell in the meristematic zone and reaches the nucleus thereof, the DNA does not usually integrate into the cell's chromosome and, thus after a few cell cycles the unintegrated heterologous DNA is lost, so that transformation by a particle gun is usually merely transient.
It would have been highly desirable to provide a method for the genetic transformation of monocotyledonous plants which would result in stable transformation with a satisfactory yield.
One of the most commercially promising monocotyledons are the Lemnaceae, a widely distributed aquatic family of small (1–5 mm) plants. The Lemnaceae excel in two characteristics potentially exploitable by the biotechnology industry: their extraordinary vegetative growth rates and a high tolerance for a spectrum of nutrients and toxic substances (Landolt and Kandeler, 1987). In the U.S.A., commercialization of Lemnaceae has centered around waste water management and animal feed (Culley et al., 1981; Ngo, 1987). However, the use of mixed aquacultures and conventional technology has met with only moderate success. A different approach was taken in Israel, utilizing the Lemna gibba Hurfeish strain (Porath et al., 1979). With its especially short root and high protein, carotenoid and iron content, this strain was cultivated under modern greenhouse conditions (4 tons harvested per acre per week; Tzora Biotechnology Inc., Kibbutz Tzora), and successfully marketed as a packaged vegetable product for the food industry. Notwithstanding the exceedingly high growth rates and the promising future of Lemnaceae as a potential food source, various attempts to genetically transform these plants, by a stable transformation method proved, to date, quite unsuccessful. The failure of transformation was due to the fact that Lemnaceae multiply vegetatively, daughter fronds arising from meristematic zones deep inside the mother frond. Thus, the meristem initial must be reached for stable transformation to take hold. Particle bombardment of Lemnaceae, the current state-of-the-art method used by several groups to obtain localized, transitory transformation events, was found by the inventors of the application ineffective in transformation of daughter fronds.
It would have been highly desirable to obtain Lemnaceae plants which are stably transformed with heterologous DNA of interest and to use such transformed plants for the production of chemical and biological products.